The invention relates to polynucleotides. More particularly, the invention relates to methods and apparatus for detecting polynucleotide hybridization in luminescence-based assays.
Polynucleotides are linear polymers composed of covalently linked nucleotides, which in turn are composed of purines (such as adenine (A) and guanine (G)), pyrimidines (such as cytosine (C), thymidine (T), and uracil (U)), carbohydrates, and phosphoric acid. Polynucleotides may be single or double-stranded. Double-stranded polynucleotides are formed of two different single-stranded polynucleotides that bind to one another through noncovalent base-pairing interactions to form a hybrid. Such hybridization will occur if the sequences of the single-stranded polynucleotides are xe2x80x9ccomplementary,xe2x80x9d so that for example wherever there is an A in one strand there is a T or a U in the other, and wherever there is a G in one strand there is a C in the other.
Polynucleotides in the form of deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) encode genetic information that controls cellular function and heredity in biological systems. DNA encodes information at least in part in the form of genes. Genes are sequences of nucleotides that encode information for constructing a polypeptide. The sequence of nucleotides in a gene may vary due to insertions, deletions, repeats, inversions, translocations, and/or single and multiple nucleotide substitutions, among others. These variations may be termed polymorphisms, and genes that differ by polymorphisms may be termed alleles.
Polymorphisms may lead to observable differences in phenotype. Familiar polymorphisms in humans include (1) variations in glycosyltransferase genes associated with the ABO blood groups, (2) variations in the apoE gene associated with Alzheimer""s disease, (3) variations in the CCR5 chemokine receptor gene associated with resistance to HIV infection, and (4) variations in the hemoglobin gene associated with sickle cell anemia.
Detection of nucleotide polymorphisms may play an important role in studies of biodiversity, evolution, and bio-identity, as well as in the understanding and treatment of disease. Detection of nucleotide polymorphisms can be facilitated using high-throughput screening (HTS). In HTS, hundreds of thousands of samples may be analyzed. Maximum efficiency in terms of speed and automation are desirable to process as many samples as possible, as rapidly as possible, and accuracy is desirable to avoid registering false positives and false negatives.
Detection of nucleotide polymorphisms also can be facilitated using luminescence assays. Luminescence is the emission of light from an excited electronic state of a xe2x80x9cluminophore,xe2x80x9d i.e., a luminescent atom or molecule. In a luminescence assay, a luminophore is included in a system, and properties of the light emitted by the luminophore are used to characterize components and properties of the system. Such light may include fluorescence and phosphorescence, and such properties may include hybridization/binding reactions and enzymatic activity, among others. In this sense, the analyte may act as a reporter to provide information about another material or target substance that is the focus of the assay. Luminescence assays may use various properties of the luminescence, including its intensity, polarization, and/or lifetime.
Luminescence polarization assays involve the absorption and emission of polarized light. (Here, polarization refers to the direction of light""s electric field, which generally is perpendicular to the direction of light""s propagation.) In a luminescence polarization assay, a luminescent sample is illuminated with polarized excitation light, which preferentially excites luminophores having absorption dipoles aligned with the polarization of the excitation light. These molecules subsequently decay by preferentially emitting light polarized parallel to their emission dipoles. The extent to which the total emitted light is polarized depends on the extent of molecular reorientation during the lifetime of the excited state, which in turn depends on the size, shape, and environment of the reorienting molecule. Thus, luminescence polarization assays can be used to quantify the extent of hybridization between polynucleotides through the effect of hybridization on the rate of reorientation.
Recently, selected luminescence assays have been used in certain genetic analyses involving microplates and the more exotic DNA chips, the latter being arrays of DNA probes or reference sequences packed together in a small area. However, these assays suffer from a number of shortcomings related in part to shortcomings in available sample holders, luminescence detection devices, and methods of background subtraction, as described below.
Shortcomings in sample holders. Microplates are a preferred sample holder in luminescence analyses. Unfortunately, despite their demonstrated utility, standard microplates suffer from a number of shortcomings. For example, sample wells in microplates and other sample holders for luminescence assays may have regions that are optically inaccessible, from which luminescence can be neither excited nor detected. Sample in such regions effectively is wasted because it does not contribute to the analysis. Wasted sample can translate into significant extra cost, particularly for assays that are performed in large numbers, that use expensive reagents, and/or that are inhomogeneous, requiring washing. Sample wells also may have walls or other regions that are themselves detectable optically, increasing background if such regions luminesce.
Shortcomings in luminescence detection devices. Photoluminescence requires illuminating a sample with light from a light source, and detecting light emitted from the sample. Photoluminescence detection devices may employ various light sources. In academic research laboratories, light sources for luminescence polarization assays have included lasers and arc lamps (e.g., xenon arc lamps). Unfortunately, these light sources suffer from a number of shortcomings. The gas in xenon arc lamps is under high pressure (about 10 atmospheres), so that explosion is always a danger. The power supplies for lasers and xenon arc lamps operate at very high currents (about 25 amps) and voltages (about 20,000 to 40,000 volts), so that electrocution and other health hazards are always a danger. In particular, the power supplies for arc lamps produce ozone and may deliver a lethal shock when the lamps are started. The power supplies also may produce transients that can damage other electronic components of the system. The light emitted by lasers and xenon arc lamps is very intense, so that eye damage is always a danger. In particular, the extreme brightness may damage the retina, and ultraviolet light emitted by xenon arc lamps and some lasers may damage the cornea. The spectral output of lasers and some (e.g., mercury) arc lamps is very limited, so that desired excitation wavelengths may not be available. The lifetime of arc lamps may be very short, typically around 300 hours, so that the lamp must be changed frequently, further exposing the operator to dangers posed by the lamp and power supply.
These shortcomings assume even greater significance outside the research laboratory. For example, in high-throughput screening applications, the light source may be used nearly continuously, so that the dangers posed by lasers and arc lamps are ever present. The light source also may be used by relatively unskilled operators, who may be unfamiliar with or unreceptive to safety issues.
In high-throughput screening laboratories, light sources for luminescence polarization assays have included incandescent (e.g., tungsten) lamps and flash lamps. Incandescent lamps are relatively common and inexpensive, and include lamps from overhead projectors. Incandescent lamps put out broad-spectrum light, so that they may be used with a variety of luminescent compounds. Flash lamps are more exotic, but provide some advantages over incandescent lamps. In particular, flash lamps may be used for both time-resolved and steady-state measurements. This flexibility allows the same light source to be used in instruments that perform multiple assays, such as steady-state and time-resolved luminescence polarization assays. Moreover, flash lamps may have long lifetimes, as long as 10,000 hours.
Shortcomings in methods of background subtraction. Optical spectroscopic assays are subject to artifacts that alter the apparent luminescence of the analyte and thus the accuracy, repeatability, and reliability of the assay. Some artifacts increase the apparent luminescence of the analyte, causing intensity-based assays to overreport the amount of light emitted by the analyte. Such artifacts include background. Other artifacts decrease the apparent luminescence of the analyte, causing intensity-based assays to underreport the amount of light emitted by the analyte. Such artifacts include scattering and absorption. Such artifacts also include changes in the composition that change the optical transfer function (photons collected/photons injected), including changes in index of refraction and surface tension.
Optical spectroscopic assays also are subject to artifacts that alter the apparent polarization of the analyte. Such artifacts also include background, scattering, and absorption, among others, and can increase or decrease the apparent polarization.
Among artifacts that alter polarization while increasing the apparent luminescence of the analyte, background is especially significant. Background refers to light and other signals that do not arise from the analyte, but that can be confused with light that does arise from the analyte. Background may arise from non-analyte luminescent components of the sample (e.g., library compounds, target molecules, etc.). Background also may arise from luminescent components of the sample container and detection system (e.g., microplates, optics, fiber optics, etc.). Background also may arise from scattered excitation light that leaks through the optical filters, which is equivalent to luminescence with a zero lifetime, and from room light.
There is no way to eliminate every source of background, so methods must be used to discriminate between analyte and background. If the analyte and background have different spectra, background may be at least partially discriminated using appropriate optical filters, which pass light emitted by the analyte but block background. If the analyte and background have overlapping spectra, background may be at least partially discriminated in two ways. First, background may be discriminated using a blank. In this method, data such as intensity data are collected for the sample and for a blank that lacks analyte but otherwise resembles the sample. Background is at least partially discriminated by subtracting the data obtained from the blank from the data obtained from the sample. Second, background may be discriminated by gating. In this method, data are collected from the sample only at times when the background is low or nonexistent.
Unfortunately, these methods of rejecting background suffer from a number of shortcomings, especially if the analyte and background have overlapping spectra. The use of blanks requires making two measurements for every sample, at least if the background is different for each sample. Background may be different for each sample if each sample is housed in a different container and/or if each sample contains a different, intrinsically luminescent target molecule, such as a peptide, protein, or nucleic acid, among others. The use of gating requires knowledge of the lifetime and intensity of the background. The use of gating also requires collecting data only over limited times, so that data collection is slowed and potentially useful data is discarded. Gating is especially problematic for short-lifetime background, because luminescence from the analyte is most intense for short times after excitation.
Among artifacts that alter polarization while decreasing the apparent luminescence of the analyte, scattering and absorption are especially significant. Scattering can arise if the composition containing the analyte is turbid, so that excitation and/or emission light are scattered out of the optical path and therefore not detected. Absorption can arise if non-analyte components of the composition can absorb excitation and/or emission light. Absorption of excitation light reduces luminescence indirectly, by reducing the amount of light available to excite luminescence. Absorption of emission light reduces luminescence directly. Collectively, absorption of excitation and emission light is termed xe2x80x9ccolor quenching.xe2x80x9d Scattering and color quenching may vary from sample to sample and therefore be difficult to characterize.
There is no way to eliminate every source of scattering and absorption. This is especially true in compositions containing biological molecules, because biological molecules such as nucleic acids and proteins may absorb light having wavelengths commonly used in luminescence assays.
Background, scattering, absorption, and other artifacts affecting apparent luminescence are significant shortcomings, even for single measurements. However, they are potentially crippling shortcomings in high-throughput genomics applications, where tens or hundreds of thousands of samples may be analyzed each day. In genomics applications, the use of blanks may double the consumption of reagents and the time required for sample preparation and data collection, as well as associated costs. Moreover, in genomics applications, biological molecules that scatter and absorb light often must be employed.
The invention provides methods and apparatus for detecting polynucleotide hybridization in luminescence-based assays.